Hyaluronic acid is a macromolecular polysaccharide without branching consisting of two kinds of monosaccharides of linearly and alternately bonded D-glucuronic acid and N-acetylglucosamine. Hyaluronic acid is present in every connective tissue of animals, and its molecular weight is considered to be from several ten thousand to several million. Hyaluronic acid is characterized by having a high moisture retention property, a high viscoelasticity and a high lubricity, and making use of such characteristics, it is blended as a humectant in cosmetics, and its availability is widely recognized in the field of medicine as e.g. an injection drug for treatment of various arthropathy or adjuncts for ophthalmic operation.
As practical methods for producing hyaluronic acid, extraction from cockscombs or umbilical cords in which hyaluronic acid is contained in a large amount, and fermentation by Streptococci which is one type of Lactobacillus have been known. Regarding enzymes which concern biosynthesis of hyaluronic acid in living organism, from research results at genetic level and enzyme level, it has been clarified that hyaluronic acid is synthesized by hyaluronan synthase from two sugar nucleotides of UDP-GlcA (uridine 5′-diphosphoglucuronic acid) and UDP-GlcNAc (uridine 5′-diphospho-N-acetylglucosamine) as precursors.
On the other hand, as an example of preparation of hyaluronic acid employing a hyaluronidase used in the present invention also, it has been reported that a pyridylaminated hyaluronic acid oligomer mixture is formed in a slight amount from a reaction system in which pyridylaminated hyaluronic acid hexasaccharide as an acceptor and hyaluronic acid having a molecular weight of 800,000 as a donor coexist, utilizing the sugar transfer activity of the hyaluronidase (The Journal of Biological Chemistry 270, 3741 (1995)). In the oligomer mixture, from pyridylaminated hyaluronic acid octasaccharide (molecular weight: about 1,600) to maximum pyridylaminated hyaluronic acid docosasaccharide (molecular weight: about 4,400) are detected even though their amounts are very small.
However, development of an enzymatic preparation means which may replace extraction from cockscombs or fermentation as a conventional method for producing hyaluronic acid has not been achieved in spite of a great number of trials.
The present inventors have conducted extensive studies on development of a novel method for producing hyaluronic acid or a hyaluronic acid derivative which is highly useful at a practical level, and as a result, unexpectedly, they have found a novel method for producing hyaluronic acid or a hyaluronic acid derivative by an enzymatic means employing a hyaluronidase for the first time.
More particularly, they have found that, in research in production of hyaluronic acid or a hyaluronic acid derivative by an enzymologic means, when a hyaluronidase which has been known as an enzyme which normally decomposes hyaluronic acid is employed as an enzyme polymerization catalyst and a hyalobiuronate oxazoline derivative is employed as a monomer substrate, the monomer substrate is enzymatically polymerized to form hyaluronic acid or a hyaluronic acid derivative having a high molecular weight with a high yield. The present invention has been accomplished on the basis of these discoveries.